Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoHI and RpoHII using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoHI and RpoHII regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the −35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoHI promoter sequence logo, is critical for RpoHI–dependent transcription; and that several bases in the predicted −10 element were important for activity with either RpoHII or both RpoH homologs. Genes that are transcribed by both RpoHI and RpoHII are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoHI regulon are associated with a classic heat shock response, while those specific to RpoHII are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoHI and RpoHII allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.

We have identified a second RpoH homolog, RpoH II , in the ␣-proteobacterium Rhodobacter sphaeroides. Primary amino acid sequence comparisons demonstrate that R. sphaeroides RpoH II belongs to a phylogenetically distinct group with RpoH orthologs from ␣-proteobacteria that contain two rpoH genes. Like its previously identified paralog, RpoH I , RpoH II is able to complement the temperature-sensitive phenotype of an Escherichia coli 32 (rpoH) mutant. In addition, we show that recombinant RpoH I and RpoH II each transcribe two E. coli 32 -dependent promoters (rpoD P HS and dnaK P1) when reconstituted with E. coli core RNA polymerase. We observed differences, however, in the ability of each sigma factor to recognize six R. sphaeroides promoters (cycA P1, groESL 1 , rpoD P HS , dnaK P1, hslO, and ecfE), all of which resemble the E. coli 32 promoter consensus. While RpoH I reconstituted with R. sphaeroides core RNA polymerase transcribed all six promoters, RpoH II produced detectable transcripts from only four promoters (cycA P1, groESL 1 , hslO, and ecfE). These results, in combination with previous work demonstrating that an RpoH I mutant mounts a typical heat shock response, suggest that while RpoH I and RpoH II have redundant roles in response to heat, they may also have roles in response to other environmental stresses.The ability to regulate gene expression through the use of alternate sigma factors enables bacteria to respond quickly to changes in the environment. These secondary sigma factors guide RNA polymerase to promoter elements whose target sequences differ from those of the primary sigma factors, thereby directing the transcription of a specific set of genes (44). One such rapid response to environmental perturbations, known as the heat shock response, uses an alternate sigma factor to increase the expression of proteins needed to maintain an intracellular milieu conducive for protein folding. While these heat-inducible gene products have traditionally been referred to as the heat shock proteins, increased heat shock protein expression is also induced by other stress conditions (11, 33). We are interested in alternate sigma factors that play a role in the heat shock or other stress responses of the ␣-proteobacterium Rhodobacter sphaeroides.In the ␥-proteobacterium Escherichia coli, 32 (encoded by rpoH) is the alternate sigma factor responsible for recognizing heat shock gene promoters (12). As a member of the 70 family of eubacterial sigma factors, 32 recognizes unique promoter sequences centered at positions Ϫ10 and Ϫ35 relative to the transcriptional start site (44). All 32 -like proteins are defined by a conserved region known as the "RpoH box"; they also contain conserved sequences in regions 2.4 and 4.2 that recognize the Ϫ10 and Ϫ35 elements, respectively, of heat shock promoters (29). While E. coli, with its single rpoH gene, sets the stage for much of what we know about bacterial heat shock regulation, several ␣-proteobacteria are known to encode multiple RpoH homologs. Three rpoH-like genes in...

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Many plants accumulate high levels of hydroxycinnamoyl esters and amides in their tissues, presumably to protect against biotic and abiotic stress. Red clover (Trifolium pretense) leaves accumulate high levels [5–15 mmol/kg fresh weight (FW)] of caffeic acid derivatives, including phaselic acid (2-O-caffeoyl-L-malate). Oxidation of caffeoyl-malate by an endogenous polyphenol oxidase (PPO) has been shown to help preserve forage protein after harvest and during storage as silage, which should improve N use efficiency in dairy and other ruminant production systems. The widely grown forage alfalfa lacks both PPO and PPO substrates and experiences substantial loss of protein following harvest. We previously identified a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT, previously called HCT2) responsible for phaselic accumulation in red clover. With the goal of producing PPO-oxidizable compounds in alfalfa to help preserve forage protein, we expressed red clover HMT in alfalfa. Leaves of these alfalfa accumulated mainly p-coumaroyl- and feruloyl-malate (up to 1.26 and 0.25 mmol/kg FW, respectively). Leaves of HMT-expressing alfalfa supertransformed with an RNA interference (RNAi) construct to silence endogenous caffeoyl-CoA acid O-methyltransferase (CCOMT) accumulated high levels of caffeoyl-malate, as well as the p-coumaroyl and feruloyl esters (up to 2.16, 2.08, and 3.13 mmol/kg FW, respectively). Even higher levels of caffeoyl- and p-coumaroyl-malate were seen in stems (up to 8.37 and 3.15 mmol/kg FW, respectively). This level of caffeoyl-malate accumulation was sufficient to inhibit proteolysis in a PPO-dependent manner in in vitro experiments, indicating that the PPO system of post-harvest protein protection can be successfully adapted to alfalfa.